7-[3,5-dihydroxy-2- (3-hydroxy-5-phenyl-pent-1-enyl)- cyclopentyl]-n-ethyl-hept-5-enamide (bimatoprost) in crystalline form ii, methods for preparation, and methods for use thereof

ABSTRACT

The present invention provides a new crystalline form of bimatoprost, designated as crystalline form II. This new crystalline form is the most stable form known to date of bimatoprost. Moreover, it has been found that bimatoprost crystalline form II is readily prepared from crystalline form I.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/263,471 filed on Nov. 23, 2009, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to crystalline forms of7-[3,5-dihydroxy-2-(3-hydroxy-5-phenyl-pent-1-enyl)-cyclopentyl]-N-ethyl-hept-5-enamide(bimatoprost) and particularly to a newly identified crystalline form ofbimatoprost. The present invention further relates to methods for itspreparation and to methods for treating disorders associated with ocularhypertension.

BACKGROUND OF THE INVENTION

Ocular hypotensive agents are useful in the treatment of a number ofvarious ocular hypertensive conditions, such as post-surgical andpost-laser trabeculectomy ocular hypertensive episodes, glaucoma, and aspresurgical adjuncts.

Glaucoma is a disease of the eye characterized by increased intraocularpressure. On the basis of its etiology, glaucoma has been classified asprimary or secondary. For example, primary glaucoma in adults(congenital glaucoma) may be either open-angle or acute or chronicangle-closure. Secondary glaucoma results from pre-existing oculardiseases such as uveitis, intraocular tumor or an enlarged cataract.

The underlying causes of primary glaucoma are not yet known. Theincreased intraocular tension is due to the obstruction of aqueous humoroutflow. In chronic open-angle glaucoma, the anterior chamber and itsanatomic structures appear normal, but drainage of the aqueous humor isimpeded. In acute or chronic angle-closure glaucoma, the anteriorchamber is shallow, the filtration angle is narrowed, and the iris mayobstruct the trabecular meshwork at the entrance of the canal ofSchlemm. Dilation of the pupil may push the root of the iris forwardagainst the angle, and may produce pupillary block and thus precipitatean acute attack. Eyes with narrow anterior chamber angles arepredisposed to acute angle-closure glaucoma attacks of various degreesof severity.

Secondary glaucoma is caused by any interference with the flow ofaqueous humor from the posterior chamber into the anterior chamber andsubsequently, into the canal of Schlemm. Inflammatory disease of theanterior segment may prevent aqueous escape by causing completeposterior synechia in iris bombe and may plug the drainage channel withexudates. Other common causes are intraocular tumors, enlargedcataracts, central retinal vein occlusion, trauma to the eye, operativeprocedures and intraocular hemorrhage.

Considering all types together, glaucoma occurs in about 2% of allpersons over the age of 40 and may be asymptotic for years beforeprogressing to rapid loss of vision. In cases where surgery is notindicated, topical b-adrenoreceptor antagonists have traditionally beenthe drugs of choice for treating glaucoma.

Prostaglandins were earlier regarded as potent ocular hypertensives;however, evidence accumulated in the last two decades shows that someprostaglandins are highly effective ocular hypotensive agents and areideally suited for the long-term medical management of glaucoma. (See,for example, Starr, M. S. Exp. Eye Res. 1971, 11, pp. 170-177; Bito, L.Z. Biological Protection with Prostaglandins Cohen, M. M., ed., BocaRaton, Fla. CRC Press Inc., 1985, pp. 231-252; and Bito, L. Z., AppliedPharmacology in the Medical Treatment of Glaucomas Drance, S. M. andNeufeld, A. H. eds., New York, Grune & Stratton, 1984, pp. 477-505).Such prostaglandins include PGF_(2α), PGF_(1α) PGE₂, and certainlipid-soluble esters, such as C₁ to C₅ alkyl esters, e.g. 1-isopropylester, of such compounds.

It is known however that many drug compounds exist in two or morecrystalline forms, referred to as polymorphs. These polymorphs of thesame molecule have identical chemical properties but may exhibitdifferent physical properties, such as melting point, solubility,hardness, etc. In such cases, the danger exists of less solublepolymorphic forms precipitating from a solution made from another moresoluble but less stable form. The formation of crystals in an ophthalmicsolution can cause serious injury to the eye. In addition, precipitationof the drug substance may cause an apparent reduction in potency andbioavailability of the product.

For these reasons, there has been interest in the polymorphic forms ofbimatoprost (currently marketed as Lumigan™). U.S. Patent ApplicationPublication No. 2009/0163596 describes bimatoprost in crystalline formI. The present invention describes a new polymorphic form ofbimatoprost.

SUMMARY OF THE INVENTION

The present invention provides a new crystalline form of7-[3,5-dihydroxy-2-(3-hydroxy-5-phenyl-pent-1-enyl)-cyclopentyl]-N-ethyl-hept-5-enamide(bimatoprost), designated as crystalline form II. This new crystallineform is the most stable form known to date of bimatoprost. Moreover, ithas been found that bimatoprost crystalline form II is readily preparedfrom crystalline form I or may be prepared directly from amorphousbimatoprost.

In another embodiment of the invention, there provided pharmaceuticalcompositions including a therapeutically effective amount of7-[3,5-dihydroxy-2-(3-hydroxy-5-phenyl-pent-1-enyl)-cyclopentyl]-N-ethyl-hept-5-enamidein crystalline form II in an ophthalmically acceptable carriertherefore.

In another embodiment, there provided methods for treating ocularhypertension Such methods can be performed, for example, byadministering to a subject in need thereof7-[3,5-dihydroxy-2-(3-hydroxy-5-phenyl-pent-1-enyl)-cyclopentyl]-N-ethyl-hept-5-enamidein crystalline form II in an ophthalmically acceptable carrier.

In another embodiment, there provided methods for treating glaucoma.Such methods can be performed, for example, by administering to asubject in need thereof7-[3,5-dihydroxy-2-(3-hydroxy-5-phenyl-pent-1-enyl)-cyclopentyl]-N-ethyl-hept-5-enamidein crystalline form II in an ophthalmically acceptable carrier.

In another embodiment of the invention there provided methods forconverting7-[3,5-dihydroxy-2-(3-hydroxy-5-phenyl-pent-1-enyl)-cyclopentyl]-N-ethyl-hept-5-enamidein crystalline form I to crystalline form II. Such methods can beperformed, for example, by

a) heating7-[3,5-dihydroxy-2-(3-hydroxy-5-phenyl-pent-1-enyl)-cyclopentyl]-N-ethyl-hept-5-enamidein crystalline form I in the solid state from about 55° C. to about 72°C. at a heating rate of about 2° C. per minute;

b) cooling the crystalline7-[3,5-dihydroxy-2-(3-hydroxy-5-phenyl-pent-1-enyl)-cyclopentyl]-N-ethyl-hept-5-enamidefrom about 72° C. to about 55° C. at a heating rate of about 0.2-0.5° C.per minute;

c) repeating steps a) and b) from 3 to about 9 times;

thereby converting crystalline form I to crystalline form II.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a characteristic DSC profile of bimatoprost in crystallineform II.

FIG. 2 is a characteristic X-ray powder diffraction (XRPD) pattern ofbimatoprost in crystalline form II.

FIG. 3 depicts thermograms of two lots of bimatoprost (Lot X10510representative of all the lots except for Lot 08-A-014-3).

FIG. 4 depicts a thermogram of a sample treatment cycle consisting ofthe partial melting and controlled cooling of Lot 08-A-014-3.

FIG. 5 depicts a thermogram of Lot 08-A-014-3 after partial melting andcontrolled cooling and demonstrates the presence of pure Polymorph II.

FIG. 6 depicts a thermogram of stress stability sample 1 of lot X10510subjected to 40° C., ambient air headspace and light exposure.

FIG. 7 depicts a thermogram of stress stability sample 2 of lot X10510subjected to 40° C., ambient air headspace and protected from lightexposure.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention claimed. As used herein, theuse of the singular includes the plural unless specifically statedotherwise. As used herein, “or” means “and/or” unless stated otherwise.Furthermore, use of the term “including” as well as other forms, such as“includes,” and “included,” is not limiting. The section headings usedherein are for organizational purposes only and are not to be construedas limiting the subject matter described.

It is to be understood that“7-[3,5-dihydroxy-2-(3-hydroxy-5-phenyl-pent-1-enyl)-cyclopentyl]-N-ethyl-hept-5-enamide”and “bimatoprost” refer to the same compound and may be usedinterchangeably throughout.

In addition, it is to be understood that “crystalline form” and“polymorphic form” may be used interchangeably throughout thespecification. “Crytalline form I” or “crystalline form II” may also bereferred to as “polymorph I” or “polymorph II”.

Unless specific definitions are provided, the nomenclatures utilized inconnection with, and the laboratory procedures and techniques ofanalytical chemistry, synthetic organic and inorganic chemistrydescribed herein are those known in the art. Standard chemical symbolsare used interchangeably with the full names represented by suchsymbols. Thus, for example, the terms “hydrogen” and “H” are understoodto have identical meaning. Standard techniques may be used for chemicalsyntheses, chemical analyses, and formulation

The present invention provides bimatoprost in a new polymorphic form,designated as polymorph II. This new, more stable polymorph wasdiscovered by exposing polymorph I to elevated temperatures andhumidities. During these studies it was found that polymorph I convertsquantitatively to polymorph II by using the controlled heating andcooling cycles set forth herein.

Bimatoprost crystalline form II was characterized using X-ray powderdiffraction (XRPD), differential scanning calorimetry (DSC), andinfrared spectroscopy.

The crystalline form II of bimatoprost exhibits a distinct XRPDspectrum, which is set forth in FIG. 1. The pattern has characteristicpeaks observed at (2θ): 3.60, 5.31, 7.09, 10.55, 12.24, 13.29, 14.55,15.85, 17.60, 18.49, 19.00, 19.65, 21.10, 22.20, 22.69, 24.75 and 26.65.

Polymorph II was determined to have an exothermic onset at about 70.1°C. and a peak at 74.1° C. in its differential scanning calorimetryprofile.

In another embodiment of the invention, there is provided a method forconverting bimatoprost polymorph I to bimatoprost polymorph II.

Conventional methods of making a desired polymorph include: modifyingcrystallization conditions (temperature, solvent, etc) stirring asuspension of polymorph I in various organic solvents (aka slurrymethod); and, adding seed crystals of polymorph II into a suspension ofpolymorph I. Solvents are used in these methods allowing sufficientmolecular motions that promote nucleation to the different polymorph.When slurry studies were conducted with bimatoprost polymorph I usingwater, ethyl acetate, or cyclohexane to produce polymorph II, the finalproduct was an oily residue. The same result was obtained when seedcrystals of polymorph II were added to the suspension. When slurriescontaining polymorph I were repeatedly heated and cooled, which is acommon technique used to promote crystallization, no crystals ofpolymorph II were produced.

The method of the invention involves heating and cooling the neat drugsubstance in the solid state. This method is unique because solvents arenot used to promote molecular mobility and subsequent nucleation.Bimatoprost polymorph I in the solid state (20 mg) was heated from 55°C. to 72° C. at a heating rate of 2° C./min followed by cooling from 72°C. to 55° C. at a rate of 0.2-0.5° C./min. The cycle was repeated 3-9times. Polymorph I was converted to polymorph II. Formation of polymorphII was confirmed by DSC and XRPD (see Examples).

Pharmaceutical compositions may be prepared by combining atherapeutically effective amount of polymorph II of bimatoprostaccording to the invention, or a pharmaceutically acceptable saltthereof, as an active ingredient, with conventional ophthalmicallyacceptable pharmaceutical excipients, and by preparation of unit dosageforms suitable for topical ocular use. The therapeutically efficientamount typically is between about 0.0001 and about 5% (w/v), preferablyabout 0.001 to about 1.0% (w/v) in liquid formulations.

For ophthalmic application, preferably solutions are prepared using aphysiological saline solution as a major vehicle. The pH of suchophthalmic solutions should preferably be maintained between 4.5 and 8.0with an appropriate buffer system, a neutral pH being preferred but notessential. The formulations may also contain conventional,pharmaceutically acceptable preservatives, stabilizers and surfactants.

Preferred preservatives that may be used in the pharmaceuticalcompositions of the present invention include, but are not limited to,benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetateand phenylmercuric nitrate. A preferred surfactant is, for example,Tween 80. Likewise, various preferred vehicles may be used in theophthalmic preparations of the present invention. These vehiclesinclude, but are not limited to, polyvinyl alcohol, povidone,hydroxypropyl methyl cellulose, poloxamers, carboxymethyl cellulose,hydroxyethyl cellulose cyclodextrin and purified water.

Tonicity adjustors may be added as needed or convenient. They include,but are not limited to, salts, particularly sodium chloride, potassiumchloride, mannitol and glycerin, or any other suitable ophthalmicallyacceptable tonicity adjustor.

Various buffers and means for adjusting pH may be used so long as theresulting preparation is ophthalmically acceptable. Accordingly, buffersinclude acetate buffers, citrate buffers, phosphate buffers and boratebuffers. Acids or bases may be used to adjust the pH of theseformulations as needed.

In a similar vein, an ophthalmically acceptable antioxidant for use inthe present invention includes, but is not limited to, sodiummetabisulfite, sodium thiosulfate, acetylcysteine, butylatedhydroxyanisole and butylated hydroxytoluene.

Other excipient components which may be included in the ophthalmicpreparations are chelating agents. The preferred chelating agent isedetate disodium, although other chelating agents may also be used inplace of or in conjunction with it.

The ingredients are usually used in the following amounts:

Ingredient Amount (% w/w) active ingredient about 0.001-5 preservative0-0.10 vehicle 0-40 tonicity adjustor 0-10 buffer 0.01-10 pH adjustorq.s. pH 4.5-7.5 antioxidant as needed surfactant as needed purifiedwater as needed to make 100%

The actual dose of the active compounds of the present invention dependson the specific compound, and on the condition to be treated; theselection of the appropriate dose is well within the knowledge of theskilled artisan.

The ophthalmic formulations of the present invention are convenientlypackaged in forms suitable for metered application, such as incontainers equipped with a dropper, to facilitate application to theeye. Containers suitable for dropwise application are usually made ofsuitable inert, non-toxic plastic material, and generally containbetween about 0.5 and about 15 ml solution. One package may contain oneor more unit doses.

Especially preservative-free solutions are often formulated innon-resealable containers containing up to about ten, preferably up toabout five units doses, where a typical unit dose is from one to about 8drops, preferably one to about 3 drops. The volume of one drop usuallyis about 20-35 ml.

The following examples are intended only to illustrate the presentinvention and should in no way be construed as limiting the subjectinvention.

EXAMPLES Screening Lots of Bimatoprost for Polymorphs

Twenty-one (21) lots of bimatoprost, representative of three evolvingmanufacturing processes, were screened for polymorphs by differentialscanning calorimetry (Perkin Elmer Thermal Analysis DSC-7). Each lot wasanalyzed in the same temperature range, from 30° C. to 85° C., atheating rates of 1.0° C. and 2.0° C. per minute. The results includedthe measured heat of fusion (ΔH), onset and peak temperature. All lotsshowed consistent results, except for one of the lots that exhibited asecond thermal transition (DSC peak) at a higher temperature (see FIG.3).

Table 1 exemplifies the results obtained and includes the lot(08-A-014-3) that yielded a different result from all other lots.

TABLE 1 Representative lots of Bimatoprost analyzed by DSC between 30°C. and 85° C. at a heating rate of 2° C./minute BimatoprostManufacturing lot# process ΔH (J/g) Onset (° C.) Peak (° C.) 91110 153.0 61.1 65.0 X10510 2 62.4 64.8 66.3 X11192 3 61.2 65.1 66.808-A-014-3 3 27.7 64.9 66.3 08-A-014-3 3 48.4 71.1 73.7Lot #X11192 is the current secondary reference standard. All lots testedshowed comparable results to the bimatoprost crystalline form I, exceptfor the one that yielded two thermal transitions. Lot #08-A-014-3 showedtwo thermal peaks, at 66.3° C. and 73.7° C. The 66.3° C. peak wassimilar to the other 20 lots of bimatoprost and is referred to aspolymorph I. The 73.7° C. peak (thermal transition) was not observed inthe other lots. The crystal form that caused the second thermaltransition is referred to as polymorph II. The identity of polymorph IIwas confirmed by additional experiments.

The Confirmation of the Identity of Polymorph II

The confirmation of the identity of Polymorph II was carried out byfollowing experiments:

A representative portion of lot 08-A-014-3 was subjected to controlledmelting in a covered DSC pan with a loosely fitting lid (to avoidpressure build-up). The sample was heated at a rate of 2.0° C. perminute to 72° C. (where all of polymorph I melted and polymorph IIpartially melted, so the melted portion was in contact with purepolymorph II solid). At that point the partially melted sample wassubjected to controlled cooling at a rate of 0.5° C. and 1.0° C. perminute (see FIG. 4).

When the sample reached 30° C., in the cooling phase, the materialpresumably would be fully crystallized as polymorph II. To confirm thishypothesis, the freshly cooled sample was scanned again at a heatingrate of 2.0° C. per minute, between 30° C. and 85° C. (see FIG. 5).Using Temperature Program 2, only one thermal transition was found inthe sample of lot 08-A-014-3 that had been subjected to TemperatureProgram 1. The increased melting point indicates that polymorph Iconverted to the higher melting (and presumably more stable) crystalstructure, polymorph II. The temperature programs described in thissection are summarized as follows:

Temperature Program 1 (Partial Melting):

-   -   1. Hold for 1.0 minute at 30° C.    -   2. Heat from 30° C. to 72° C. at 2.0° C./minute    -   3. Hold for 1.0 minute at 72° C.    -   (note: 73-74° C. is the apex of the peak representing the        melting of Polymorph II)    -   4. Cool from 72° C. to 55° C. at 0.5° C./minute    -   5. Cool from 55° C. to 30° C. at 1.0° C./minute

Temperature Program 2 (Complete Melting):

-   -   1. Hold for 1.0 minute at 30° C.    -   2. Heat from 30° C. to 85° C. at 2.0° C./minute        The results are shown in Table 2.

TABLE 2 Results of lot 08-A-014-3 after Temperature Programs 1 and 2Sample Onset (° C.) Peak (° C.) Polymorph I (08-A-014-3) 65.4 66.8Polymorph II (08-A-014-3) 71.9 74.4

The lot (08-A-014-3) that was subjected to the DSC temperature programsspecified above was analyzed by HPLC, and the chromatographic resultswere compared to those obtained from the control sample (stored at −20°C.) of the same lot. The chromatographic comparison showed that thesample subjected to a partial melting followed by a complete melting wasintact bimatoprost having an impurity profile essentially the same asthat of the control (frozen) sample.

Since polymorph II was confirmed and identified as bimatoprost,subsequently released bimatoprost lots were also analyzed by DSC. Theresults showed that only polymorph I was contained in these lots.

Bimatoprost samples representing fourteen (14) lots were submitted forX-ray powder diffraction analysis to SSCI, Inc. (3065 Kent Avenue, WestLafayette, Ind. 47906). According to SSCI analysis, the results showedthat the patterns of all samples, except lot 08-A-014-3, were similar toeach other in terms of peak positions, suggesting that these sampleswere the same morphological form. The pattern of lot 08-A-014-3contained additional reflections, suggesting that this sample was eithera mixture of forms or a second crystalline form. The DSC and HPLCresults support the conclusion that lot 08-A-014-3 was a mixture of thetwo polymorphic forms of bimatoprost.

Recrystallization of Bimatoprost

Bimatoprost lot #X11113 was used in the recrystallization study. It wasrecrystallized using seven solvent systems:

1 dichloromethane/hexane2 chloroform3 chloroform/toluene4 chloroform/hexane5 ethyl acetate6 dichloromethane7 chloroform/pentaneA sample isolated from each solvent system was analyzed by DSC. Theresults showed the exclusive presence of polymorph I in all samples.HPLC assays were also done for each recrystallized sample, and the assayresults are shown in Table 3.

TABLE 3 HPLC Assay Results of Recrystallized Bimatoprost SamplesImpurity (area %) Sample recrystallized Bimatoprost Total 5,6-trans15-keto from system indicated (% w/w) unspecified 15-β isomer isomeranalog X11113 control 98.17 0.04 0.44 0.28 0.22 dichloromethane/hexane97.76 0.16 0.20 0.21 0.40 chloroform 97.06 0.40 0.27 0.25 0.76chloroform/toluene 97.63 0.50 0.32 0.16 0.41 chloroform/hexane 97.560.43 0.24 0.23 0.81 ethyl acetate 97.44 0.22 0.25 0.22 0.53dichloromethane 97.78 0.24 0.21 0.21 0.40 chloroform/pentane 97.39 0.290.29 0.22 0.55The chromatographic results of the recrystallized samples werecomparable to those of the corresponding controls (beforerecrystallization). An increase in the 15-keto levels was the likelyresult of exposure of the samples to ambient air during therecrystallization experiments. No attempt was made to protect thesamples from ambient conditions (temperature, air, humidity, light)during the recrystallization experiments. The bimatoprost assay valuesremained above 97% w/w, and the overall results indicated that therecrystallization process did not substantially degrade the samples.

Solid-State Polymorphic Transformation in Samples of the FormalStability Program

Upon routine visual examination, one of the Bimatoprost samples (lotX10510) in formal stability showed an amber-colored particle embedded ina larger rock of white active pharmaceutical ingredient (API). DSC andIR analyses confirmed that sample X10510 stored 18 months at 25° C./60%RH yielded substantial amounts of polymorph II. The colored particle wasseparated from the white drug substance, and DSC was performed on both.The results are shown in Table 4.

TABLE 4 Calorimetric results of the white and the colored Bimatoprostparticles found in lot X10510 at 25° C./60% RH (at 18 months) ParticleΔH (J/g) Onset (° C.) Peak (° C.) White 75.6 70.2 73.0 Colored 73.3 70.473.4

The particles were assayed by HPLC and the results confirmed that theywere intact bimatoprost. The same samples were submitted for solid-stateIR spectral analysis. The results of the spectroscopic investigationconfirmed that the white and colored particles had no substantialchemical or morphological differences. A comparison of the spectra ofthese samples with that of the control indicated some distinctdifferences in crystal habit.

In order to determine whether bimatoprost spontaneously converts frompolymorph I to polymorph II at elevated temperatures (i.e. higher thanthe prescribed storage temperature), a number of other lots in theformal stability program were also analyzed by DSC. The results aresummarized in Table 5.

TABLE 5 Calorimetric results of representative samples in the formalstability program Time Sample Stability point Particle ΔH Onset Peak lotcondition (months) type (J/g) (° C.) (° C.) X10510 40° C./75% RH 3 white69.2 67.4 71.5 X11113 40° C./75% RH 3 white 67.0 69.3 72.5 X10554 25°C./60% RH 18 white 79.8 70.6 73.1 translucent 78.5 70.9 73.9All the formal stability samples tested consisted of polymorph II. Sincethe control (frozen) samples from the same lots were previously analyzedby DSC with the results showing that none of them contained polymorphII, there is a clear indication that a polymorphic transformation hadoccurred under the stability conditions employed.

Solid-State Polymorphic Transformation in Laboratory Stress StudySamples

Spontaneous solid-state polymorphic conversion was observed in alaboratory stress stability study. Two Bimatoprost lots (X11192 andX10510) were subjected to a variety of experimental stress conditions at40° C. (see Table 6). The experimental design included ambient air andargon headspaces, exposure to fluorescent light, and differentsurface-to-volume ratios. The 70-day time-point samples were analyzed byHPLC and DSC. The DSC results show that all the samples were polymorphI, except for two samples of lot X10510 that were subjected to ambientair headspace (regardless of light exposure). These samples underwent apartial morphological conversion in which both polymorphs I and II werepresent simultaneously (see FIGS. 6 and 7).

TABLE 6 Calorimetric results of the laboratory stress stability sampleswith the lower surface-to-volume ratio (4 mm layer thickness) frombimatoprost lots X11192 and X10510 Sample Storage Conditions ΔH (J/g)Peak (° C.) Peak (° C.) X11192 Control (freezer) 61.2 66.8 — X11192 40°C./air/light 60.4 66.9 — X11192 40° C./argon/light 60.2 66.9 — X1119240° C./air/dark 61.3 67.1 — X11192 40° C./argon/dark 60.8 67.2 — X10510Control (freezer) 62.4 66.3 — X10510 40° C./air/light 58.0* ~63 71.8X10510 40° C./argon/light 56.6 65.0 X10510 40° C./air/dark 59.2* 65.473.0 X10510 40° C./argon/dark 58.2 63.6 — *Total heats of fusion of bothpolymorphs I and IIThe HPLC results indicate that the samples that showed two thermaltransitions were intact bimatoprost and not a degradation product. Threesamples were analyzed by infrared spectroscopy (IR), NMR and XRPD tofurther confirm the identity of each sample. These samples were asfollows:Sample A X10510 API stored at 40° C./75% RH for 3 months; polymorph IISample B X10510 API stored at 40° C./light/air headspace for 70 daysSample C X10510 API control sample stored in freezer (see Table 6);polymorph I

NMR characterization of the samples confirmed that all were intactbimatoprost.

The examination by IR spectroscopy also confirmed that the samples wereintact bimatoprost. The differences in the spectra revealed that SamplesA and C represented different crystal habits. The IR spectrum of SampleB suggests that the crystal habit is a mixture of Samples A and C.Accordingly, DSC results show that Sample B is partially converted topolymorph II.

Determination of the Aqueous Solubility of Polymorph II

The aqueous solubility of polymorph II was determined by preparing induplicate a 0.4% suspension of a formal stability sample (lot X10510stored at 25° C./60% RH for 23 months, confirmed as polymorph II).Temperature Program 1 (hold for 1.0 minute at 30° C. and then heat from30° C. to 85° C. at 2.0° C. per minute), was used to test the 23-monthstability sample by DSC to reconfirm the presence of only polymorph II(which was, indeed, the case). The suspension was allowed to rotatecontinuously over a weekend. The supernatants were assayed by HPLC usingX11192 Secondary Reference Standard to determine the drug contents. Thesolubility of polymorph II of bimatoprost was found to be 0.3% w/w,which also coincides with the solubility of polymorph I. Thus, there isno difference in terms of aqueous solubility between the two polymorphs.Since the active pharmaceutical ingredient will be used in a formulationat a concentration that is only one-tenth of the aqueous solubility ofthe active pharmaceutical ingredient (as either polymorph), there is noeffect of crystal habit on product characteristics or manufacturingprocess.

CONCLUSIONS

The confirmed polymorphism of bimatoprost has no effect on thephysicochemical stability of the liquid formulation, the drug product.The solubility of either polymorph is ten times higher than the drugconcentration in the product. No polymorphic conversion or degradationwas observed in any of the lots that were stored under the recommendedstorage conditions.

While this invention has been described with respect to these specificexamples, it is understood that other modifications and variations arepossible without departing from the spirit of the invention.

1.7-[3,5-dihydroxy-2-(3-hydroxy-5-phenyl-pent-1-enyl)-cyclopentyl]-N-ethyl-hept-5-enamidehaving the structure

in crystalline form II.
 2. A crystalline form of7-[3,5-dihydroxy-2-(3-hydroxy-5-phenyl-pent-1-enyl)-cyclopentyl]-N-ethyl-hept-5-enamidehaving an X-ray powder diffraction pattern with peaks at about (2θ):3.60, 5.31, 7.09, 10.55, 12.24, 13.29, 14.55, 15.85, 17.60, 18.49,19.00, 19.65, 21.10, 22.20, 22.69, 24.75 and 26.65.
 3. The crystallineform of claim 1 having the X-ray diffraction pattern substantially asshown in FIG.
 2. 4. The crystalline form of claim 1 having an exothermiconset at about 70.1° C. and a peak at 74.1° C. in its differentialscanning calorimetry profile.
 5. The crystalline form of claim 1 havingthe DSC profile as shown in FIG.
 1. 6. A pharmaceutical compositioncomprising a therapeutically effective amount of7-[3,5-dihydroxy-2-(3-hydroxy-5-phenyl-pent-1-enyl)-cyclopentyl]-N-ethyl-hept-5-enamidecrystalline form II in an ophthalmically acceptable carrier therefore.7. A method for treating ocular hypertension comprising administering toa subject in need thereof7-[3,5-dihydroxy-2-(3-hydroxy-5-phenyl-pent-1-enyl)-cyclopentyl]-N-ethyl-hept-5-enamidein crystalline form II in an ophthalmically acceptable carrier.
 8. Themethod of claim 7 wherein the ophthalmically acceptable carrier isselected from the group consisting of ophthalmically acceptablediluents, buffers, hydrochloric acid, sodium hydroxide, preservatives,stabilizers, tonicity adjustors, viscosity-enhancing agents, chelatingagents, surfactants and/or solubilizers and combinations thereof.
 9. Amethod for treating glaucoma comprising administering to a subject inneed thereof7-[3,5-dihydroxy-2-(3-hydroxy-5-phenyl-pent-1-enyl)-cyclopentyl]-N-ethyl-hept-5-enamidein crystalline form II in an ophthalmically acceptable carrier.
 10. Thecrystalline form of claim 1 substantially free of other crystallineforms.
 11. A method for converting7-[3,5-dihydroxy-2-(3-hydroxy-5-phenyl-pent-1-enyl)-cyclopentyl]-N-ethyl-hept-5-enamidein crystalline form I to crystalline form II comprising: a) heating7-[3,5-dihydroxy-2-(3-hydroxy-5-phenyl-pent-1-enyl)-cyclopentyl]-N-ethyl-hept-5-enamidein crystalline form I in the solid state from about 55° C. to about 72°C. at a heating rate of about 2° C. per minute; b) cooling thecrystalline7-[3,5-dihydroxy-2-(3-hydroxy-5-phenyl-pent-1-enyl)-cyclopentyl]-N-ethyl-hept-5-enamidefrom about 72° C. to about 55° C. at a cooling rate of about 0.2-0.5° C.per minute; c) repeating steps a) and b) from 3 to about 9 times;thereby converting crystalline form I to crystalline form II.